The present invention relates generally to gene delivery, and more specifically, to the preparation and use of bacteriophages modified with ligands to deliver genes that alter the phenotype, function, gene expression, or viability of a cell in a therapeutic manner.
In one approach, gene therapy attempts to target cells in a specific manner. Thus, a therapeutic gene is linked in some fashion to a targeting molecule in order to deliver the gene into a target cell or tissue. Current methods typically involve linking up a targeting molecule such as a ligand or antibody that recognizes an internalizing receptor to either naked DNA or a mammalian cell virus (e.g. adenovirus) containing the desired gene. When naked DNA is used it must be condensed in vitro into a compact geometry for entry into cells. A polycation such as polylysine is commonly used to neutralize the charge on DNA and condense it into toroid structures. This condensation process, however, is poorly understood and difficult to control, thus, making the manufacturing of homogeneous gene therapy drugs extremely challenging.
Mammalian viruses, in contrast, can package DNA into uniform particles, but, because of their complexity, they are difficult to genetically manipulate and the manufacture of viral particles for gene delivery is costly and time-consuming. Bacteriophages offer an attractive alternative as a natural method for condensing and packaging therapeutic DNA and, because of their simplicity, are relatively easy to genetically manipulate (and retain function). Moreover, because bacteriophage are extremely simplistic entities, large-scale production of phage-based gene-delivery vectors would be easier and less expensive than the production of mammalian viral vectors, for example.
Bacteriophage, such as lambda and filamentous phage, have occasionally been used in efforts to transfer DNA into mammalian cells. In general, transduction of lambda was found to be a relatively rare event and the expression of the reporter gene was weak. In an effort to enhance transduction efficiency, methods utilizing calcium phosphate or liposomes (which do not specifically target a cell surface receptor) were used in conjunction with lambda. Gene transfer has been observed via lambda phage using calcium phosphate co-precipitation (Ishiura, M. et al, Mol. Cell. Biol., 2: 607-616, 1982), or via filamentous phage using DEAE-dextran or lipopolyamine (Yokoyama-Kobayashi and Kato, Biochem. Biophys. Res. Comm. 192: 935-939, 1993; Yokoyama-Kobayashi and Kato, Anal. Biochem. 223: 130-134, 1994). However, these methods of introducing DNA into mammalian cells are not practical for gene therapy applications, as the transfection efficiency tends to be low, non-specific, and transfection is not only cumbersome, but is promiscuous regarding cell type. More reliable means of targeting vectors to specific cells (or receptors) and of guaranteeing a therapeutically useful degree of gene delivery and expression are thus required, if bacteriophage are to be shaped into vectors useful in therapeutic applications.
Attempts to target filamentous phage to cells using a fusion of a cyclic RGD peptide and a phage coat protein or a peptide-coat protein fusion have met with limited success. Although the phage are targeted and internalized, phage gene expression was neither expected nor reported (Hart et al., J. Biol. Chem. 269: 12468-12474, 1994; Barry et al., Nature Med. 2: 299-305, 1996). While it is generally understood that the RGD peptide sequence used by Hart et al. binds to integrins, Hart describes RGD mediated uptake of phage as a process similar to phagocytic uptake of bacteria via the protein invasin (an RGD protein); adenoviruses use RGD-integrin binding in conjunction with ligand-receptor binding for internalization. It is therefore not clear that RGD-integrin binding facilitates the entry of the peptide or fusion protein via a receptor mediated-endosomal mechanism, a mechanism which has been shown to yield superior results.
Thus, for gene delivery applications, methods and therapeutic agents that are simple to perform and manufacture, efficient, and target to specific cells would be very beneficial. Similarly, vectors that deliver therapeutically useful quantities of genes of interest via numerous routes of administrationxe2x80x94including oral meansxe2x80x94would be desirable. In response to these long-felt needs, the present invention provides compositions and methods for gene delivery using bacteriophage that express a ligand and carry a gene of interest, as well as provide other related advantages.
In one aspect, the invention provides a method of gene delivery, comprising: contacting a mammalian cell with filamentous phage particles presenting a ligand on their surfaces, wherein a vector within the phage encodes a gene product under control of a promoter.
In related aspects, the invention provides methods of treating tumors, smooth muscle cell diseases, or angiogenic diseases, comprising administering a pharmaceutical composition to a patient comprising a physiologically acceptable buffer and filamentous phage particles presenting a ligand on their surfaces, wherein the phage genome encodes a therapeutic gene product under control of a promoter.
In a preferred embodiment, the ligand is a polypeptide reactive with FGF receptor, and in a most preferred embodiment the ligand is FGF-2. In other embodiments, the ligand is an antibody and preferably a single-chain antibody.
The ligand may be genetically fused with a phage capsid protein or chemically conjugated to form a covalent attachment or by sandwich method. Generally, the capsid protein for gene fusion is gene III or gene VIII.
In a preferred embodiment, an endosomal escape moiety is incorporated into the ligand or displayed by other means on the surface of the phages. The ligand or phage may also further comprise a nuclear localization sequence.
In another preferred embodiment, the phage genome is a phagemid.
In preferred embodiments, the therapeutic gene product is selected from the group consisting of protein, ribozyme, and antisense oligonucleotide, and in other embodiments the therapeutic gene product is a cytotoxic agent (e.g., ribosome inactivating protein, such as saporin) or is an antibody that binds to HER2/neu.
In other aspects, the invention provides a pharmaceutical composition comprising a physiologically acceptable buffer and filamentous phage particles presenting a ligand on their surfaces, wherein the phage genome encodes a therapeutic gene product under control of a promoter and filamentous phage particles presenting a ligand on their surfaces, wherein the phage genome encodes a therapeutic gene product under control of a promoter.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.